Cloning And Characterization Of A Novel Activin Receptor-interacting Protein 4

Activin is a member of the transforming growth factorβ(TGF-β) family of growth and differentiation factors and plays important roles in the early development of embryo, neurogenesis, hematopoiesis, apoptosis and hormone secretion from new born to adult. Two types of activin receptors have been identified, type I and type II, which belong to the family of serine/threonine kinase receptors. The type II receptors bind specifically to activin and then recruit the type I receptors. Subsequently, the specific signal is translocated to the nucleus via cascade responses of Samds. Effect of activin on target cells is tissue specific, which is possibly associated with the difference in intracellular signal transductions. Recently, a group of PDZ proteins had been identified as upstream molecules of Smads in activin-mediated signal pathway and they could interact with activin type IIA receptor (ActRIIA) named activin receptor-interacting proteins (ActRIPs). Interestingly, ActRIPs are different in tissue distribution pattern, expression style and biological effects, thus, they may play a key role in determining the activin tissue sepicifity. Identification of ActRIPs contributes to better understanding the regulatory mechanisms of serine/threonine kinase receptors-mediated signaling pathway. In this study, the characterization of ActRIP4 was further investigated based on the identification of its gene. Roles of ActRIPs in activin signaling responses were also described through comparing the differences of ActRIPs in tissue distribution and regulation effects on AcTRII expression, as well as signaling transduction. These findings reveal that ActRIPs are key signal regulators in activin-mediated tissue specific responses and provide also the theory and experiment basis for investigating activin-relatated physiology, pathology and disease.Part I: Cloning and structure analysis of ActRIP4In the study of cytoplasmic proteins interacting with activin receptors, a novel cDNA clone encoding the full-length ActRIP4 was obtained from a mouse brain cDNA library. ActRIP4 gene encodes a 118 amimo acid with an NH2-terminal PDZ domain (GLGF core peptide), which is considered essential for the protein-protein interactions. There is a PDZ domain in the NH2 - terminal region of ActRIP4, ActRIP 2 and ActRIP 3, while there are five PDZ domains in the ActRIP1, which only PDZ5 has ability to interact with ActRIIA. Amino acid sequence analysis reveals that the PDZ domain of ActRIP4 is 30% homologous with the PDZ5 of ActRIP1, and 72% homologous with ActRIP2. Both of ActRIP 1-3 identified previously can combine with ActR IIA by its ActR II -binding domain (containing GLGF core peptide) and mediate activin signal transduction, suggesting that ActRIP4 may have the ability to bind specifically ActR II A..Part II: Interaction of ActRIP4 with ActR IIATo determine whether ActRIP4 can bind ActRIIA in mammalian cell or not, mammalian two-hybrid assay was performed in CHO cell. Luciferase activity of the cell co-transfected with pACT-ActRIP4 and pBIND-ActRIIA was significantly higher than that of the cell transfected only with pACT-ActRIP4 or pBIND-ActRIIA, indicating that ActRIP4 is capable of interacting with ActRIIA. Immunoprecipitation assay confirmed that ActRIP4 was able to interact stably with ActRIIA,To further investigate the effect of ActRIP4 on activin signal transduction, activin-mediated specific gene transcription activity has been determined in the HEK293 cell line co-transfected with CAGA-lux and pcDNA3-FLAG-ActRIP4. Our previous studies showed that ActRIP2 could inhibit the activin-mediated specific gene transcription activity in does-dependent manner. In contrast, the data demonstrated that ActRIP4 can enhances the transcription activity in does-dependent manner. These results suggested that ActRIP2 and ActRIP4 could have different bioactivity in signal regulation, thus co-expressions could modulate activin-mediated signal transduction in opposite directions.In addition, ActRIP4 could increase directly the ActRIIA expression on cell member. Previous studies had reported ActRIP1 could regulate the activin signal transduction via ActRIIA-ActRIPl-Smad3 pathway, ActRIP2 could down-regulate activin signal transduction through enhancing endocytosis, and ActRIP3 could up-regulate activin signaling transduction via increasing ActRIIA mRNA expression.Part III: Comparison of ActRIP4 and ActRIP1-3 expressions in mouse tissuesSince activin has significant tissue specificity, to determine whether the tissue distribution of ActRIPs is associated with activin functions or not, the tissue distribution of ActRIP4 and ActRIP1-3 in mice was analysis. By the results of RT-PCR assay, ActRIP4 mRNA distributes widely in various tissues such as heart, brain, kidney, liver, testis. ActRIP4 mRNA was mainly expressed in heart, brain, kidney and liver. In contrast, ActRIP1 mRNA was only expressed in nervous tissue.Therefore, ActRIP4, a positively regulatory protein, is different from ActRIP1-3 in molecular structure, tissue distribution, as well as biofunctions and patterns.Part IV: Bioactivities of ActRIP4 in macrophagesActivin has been demonstrated that it can independently increase the NO secretion from macrophage cell line RAW264.7 cells. Contrary to that of ActRIP2, the up-regulation of ActRIP4 on activin-mediated NO secretion was also detected by gene overexpression assay. Furthermore, the phagocytosis can be enhanced by overexpression ActRIP4 in does-dependent manner, but not ActRIP2. Thus, ActRIP2 and ActRIP4 may have antagonistic effects on activin mediated responses and a balance between positive and negative factors regulates activin-mediated gene expression and inhibition.In conclusion, ActRIP4 takes part in the regulation of ActRIIA gene expression via interaction between PDZ-like domain in its N- terminal region and ActRIIA. ActRIP4, as a novel member of ActRIP family, is distinct from ActRIP 1-3 in tissue distribution and bioactivity, resulting in regulating the activin-mediated specific signaling transduction.Part V . The mechanisms of ActRIP4-mediated differentiation of macrophage line RAW264.7 cells induced by activin to DCTo compare the biologic functions between LPS and activin, the expressions of ActRIPs, IL-1 and iNOS, as well as the phagocytosis were analyzed in RAW264.7 cells stimulated with LPS or activin. The results showed that activin had negative effects on LPS. Moreover, activin can block the Toll like receptor-4 expression on the surface of RAW264 cells in does-dependent manner using Flow cytometric analysis and the changes of molecular expression on the macrophages stimulated by LPS.In addition, mature dendritic cell (DC) could high express the CD80,CD86,MHC I , MHC II expression on cell membrane. CD14 and CD68 are identification marker of mature macrophages. To further investigate that activin played roles in RAW264.7 differatiation to dendritic cell. The same times, the up-regulation of ActRIP4 on CD80, CD86, MHC I , MHC II expression and IL-12 secretion was confirmed by ActRIP4 overexpression, moreover down-regulation of CD14 expression. These findings suggest that ActRIP4 is a key regulator contributing in specific signaling transduction of activin.